Abnormalities in caveolae and lung endothelial cell (ECs) function may result in alterations of lung vascular permeability. This may be a key element of the complex pathophysiology underlying acute lung injury. The understanding of the molecular mechanisms that regulate caveolae-mediated transcytosis and its role in mediating increased lung microvessel endothelial permeability are unclear. Our Supporting Data suggest that the crucial scaffold protein in lung ECs, intersectin-1s (ITSN-1s), plays an important role in caveolae trafficking through its interaction with dynamin and other key proteins regulating endothelial permeability. Our Supporting Data suggest that ITSN-1s is crucial in recruiting dynamin at the neck region of caveolae and that dynamin presence at the endocytic site and its ability to hydrolyze GTP are essential for caveolae release from the plasma membrane during caveolae transcytosis. However, the relationship between ITSN-1s and dynamin and its significance in the mechanism of increased lung vascular endothelial permeability are not yet defined. Strikingly, ECs deficient in ITSN-1s became apoptotic by activation of the mitochondrial death pathway through a mechanism that involves inactivation of the survival kinase Erk1/2. Thus, besides regulating transcytosis, ITSN-1s is also an anti-apoptotic protein that may have consequences in mediating lung microvascular injury secondary to apoptosis of ECs. The central hypothesis of the proposed studies is that ITSN-1s is a pivotal protein mediating the cross-talk between caveolae endocytosis and signaling pathways that regulates lung vascular endothelial permeability and EC survival. The following specific aims will test this hypothesis: Specific Aim 1 will define the role of ITSN-1s, via its SH3 domains, as a regulator of dynamin function. Specific Aim 2 will address the functional significance of ITSN-1s/dynamin interaction in the mechanism of caveolae release from the plasma membrane of lung microvessel ECs and in regulating lung microvascular permeability in mice. Specific Aim 3 will delineate the signaling mechanisms controlled by ITSN-1s that defend lung endothelium against apoptotic cell death and lung vascular endothelial injury. With the successful completion of the aims, we hope to provide novel insights into the i) pivotal role of the SH3 domains of ITSN-1s in regulation of dynamin function in lung microvessel ECs, ii) functional significance of ITSN/dynamin interaction in caveolae endocytosis and lung endothelial barrier function, and iii) signaling pathways and mechanisms governing the anti-apoptotic effects of ITSN-1s in ECs and its pathophysiological consequence in the mechanism of lung microvascular injury. We hope to provide a novel understanding of transcytosis and its regulation of lung vascular endothelial permeability and specific molecular therapeutic targets directed against inappropriate leakiness of the lung microvascular barrier. This study will provide novel insights regarding i) the role of intersectin-1s in regulation of lung vascular endothelial permeability and ii) the signaling pathways and mechanisms governing the anti-apoptotic effects of intersectin-1s in endothelial cells. Confirming intersectin-1s as a link between microvascular permeability, apoptosis and lung disease, we may find new targets against inappropriate leakiness of the lung microvascular barrier and slow the alveolar destruction associated with acute respiratory distress syndrome (ARDS) and acute lung injury (ALI).